Method of separating bacteria from free living amoebae

ABSTRACT

New protozoan derived microbial consortia and method for their isolation are provided. Consortia and bacteria isolated therefrom are useful for treating wastes such as trichloroethylene and trinitrotoluene. Consortia, bacteria isolated therefrom, and dispersants isolated therefrom are useful for dispersing hydrocarbons such as oil, creosote, wax, and grease.

The United States Government has rights in this invention pursuant tocontract no. DeAC05-84OR21400 between the United States Department ofEnergy and Martin Marietta Energy Systems, Inc.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 07/693,998,filed Apr. 26, 1991, abandoned.

FIELD OF THE INVENTION

This invention relates to microbial consortia, and methods for alteringor degrading wastes and contaminants. More particularly, this inventionrelates to protozoan derived consortia comprised of protozoa andbacteria, methods for isolating protozoa/bacteria consortia, methods forusing protozoa/bacteria consortia for altering or degrading wastes andcontaminants, and production and use of dispersants derived fromprotozoa/bacteria consortia.

BACKGROUND OF THE INVENTION

There is a need to alter or degrade solutions of waste and contaminants.In order to protect and remedy the increasingly polluted ecologicalsphere while continuing to make industrial and technological progress,it is necessary to provide effective means for altering or degradingchemical and biological wastes. To alter a substance is to chemicallychange the substance in some way; to degrade a substance is to alter thesubstance by breaking down the molecular structure thereof.

Trichloroethylene (TCE) is a prevalent chemical waste which has enteredthe environment at many Environmental Protection Agency (EPA) Superfundsites. These compounds are suspected carcinogens, and, being resistantto aerobic degradation, threaten water supplies.

Conventional techniques used to remedy contaminated sites are fraughtwith difficulty. Chemical treatment of high volume, contaminated watersuch as hexane extraction is not cost effective. Air stripping, whilelower in cost, merely dilutes the pollutant into the air. Thus, aneffective, low cost biological treatment method would be a significantstep forward in remediation of contaminated sites. TCE is degraded by avariety of mechanisms. In anaerobic environments, TCE may be convertedto more potent carcinogens such as vinyl chloride. TCE biodegradation byaerobic consortia or pure cultures of methanotrophs and pseudomonads hasalso been reported. Toluene dioxygenase enzyme from pseudomonads hasbeen shown capable of TCE alteration, or degradation. However, eithertoluene or phenol was required. Other methanotrophic cultures candegrade TCE, apparently by the methane monoxygenase enzyme, withoutadded toluene.

In various studies of biodegradation of a variety of toxic chemicalsseveral problems were apparent. Some of the environments were apparentlytoxic to the test microbes or the chemicals were adsorbed to the soilparticles and not available for degradation by the bacteria. Also, somesoils contain extremely small particles, i.e. "fines" which preventbacteria from penetrating between them and degrading the ensconcedchemical(s).

Bacteria used to degrade TCE and other toxic chemicals are currentlyisolated directly from environmental sources.

OBJECTS OF THE INVENTION

Accordingly, it is an object of the present invention to provide new anduseful microbial consortia.

It is another object of the present invention to provide new andimproved methods for altering and degrading wastes and contaminants.

It is a further object of the present invention to provide newbiologically derived compositions for chemically altering wastes andcontaminants.

Further and other objects of the present invention will become apparentfrom the description contained herein.

SUMMARY OF THE INVENTION

In accordance with an aspect of the invention, a method of separating aheterotrophic bacterium from a free living amoeba/bacteria consortiumgenerally comprises the following steps:

providing a source of a free living amoeba/bacteria consortium, theamoeba/bacteria consortium being essentially identical to at least oneof American Type Culture Collection Deposit Numbers 55120, 40908, 55342,or 55343;

placing at least a portion of the source in contact with mineral saltsagar having thereon live E. coli as a food source to contact the freeliving amoeba with the mineral salts agar and with the live E. coli;

incubating the free living amoeba/bacteria consortium in air to producea migratory growth of the amoeba/bacteria consortium;

incubating at least a portion of the migratory growth in methane in airto produce further growth of the amoeba/bacteria consortium; and,

transferring at least a portion of the further growth to a medium whichis selective for heterotrophic bacteria to separate the heterotrophicbacterium from the microbial consortia growth.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Free-living amoebae populations were first isolated, and bacteria ofinterest were then subsequently isolated from the amoebae populations.The process of selecting initially for protozoa resulted in theisolation of microbial cultures capable of altering or degrading toxicor hazardous wastes. Some of the cultures produced dispersants.

Amoebae/bacteria consortia were discovered and isolated as follows.Water samples were obtained from several wells which were used tomonitor a waste disposal site near Oak Ridge, Ten. The test site wasused previously for dumping of a variety of organic solvents includingtrichloroethylene (TCE). The number, depth, and approximateconcentrations of TCE of the test wells were as follows: well 14, 13ft., 2,100 ppb; well 27, 30 ft., 13,000 ppb; and well 46, 20 ft., 230ppb. Water samples were aseptically collected by a nitrogen displacementsampling device (Well Wizard 3013; Q.E.D. Environmental Systems, Inc.,Ann Arbor, Mich.). Samples were collected only after the well lines hadbeen cleared through several cycles of pumping. The water samples werefiltered through 1.2 μm cellulose nitrate/acetate filters (MilliporeCorp., Bedford, Mass.) which were then inverted and placed on mineralsalts (NATE) agar. Prior to the addition of the filter pads, the plateswere spread with a lawn of live E. coli. The test plates were incubatedin air at room temperature (23°-25° C.) for 7-14 days. When amoebicoutgrowths had migrated to the edge of the petri dishes the plates weretransferred to desiccator jars flushed with 10% methane in air. Theresultant microbial consortia, given identification numbers 14, 27b,27p, and 46, appeared after 2 weeks in the methane atmosphere along thearea of amoebic outgrowth. The consortia were aseptically transferredevery 3-4 weeks onto NATE agar media plates incubated in a methane inair atmosphere. Heterotrophic bacteria from the consortia were isolatedand maintained at room temperature on trypticase soy agar (TSA).

Water from all three test wells yielded free-living amoebae on the NATEplates spread with E. coli as a food source. Bacterial growth occurredin the methane atmosphere along the area of amoebic migration,demonstrating that amoebae could harbor methanotrophic bacteria. Controlplates without E. coli, which allows for amoebic migration, did notsupport methanotrophic growth away from the filter. Bacterial growthsubsequently occurred on transfer of the amoebic populations to freshNATE plates without added E. coli in a methane atmosphere. Theseconsortia can apparently be maintained indefinitely by subculture onNATE in a methane atmosphere.

Individual components of the consortia were isolated and characterized.Heterotrophic and methanotrophic bacteria and amoebae continuallycoexist in the consortia. Microscopic examination of the amoebictrophozoites and cysts indicated they were Hartmannella. The presence ofheterotrophs in these consortia grown in a methane atmosphere wasevident on transfer of aliquots from NATE to TSA. Microscopic andenzymatic analysis showed the resultant heterotrophic populations were amixture of genera, including but not limited to Pseudomonas,Alcaligenes, Bacillus, Moraxella, Cytophaga, Paracoccus andHyphomicrobium, as shown in Tables 1, 2, and 3.

When suspensions of the consortia were filtered through a 0.8 μm filterand the filtrate subcultured three times on TSA the resultantheterotrophs could no longer grow when replated on NATE in a methane inair atmosphere. Conversely, when amoebic populations from the NATEplates in a methane atmosphere were subcultured three times in air onnonnutrient agar spread with a lawn of live E. coli (NNAE) plates andthe newly generated peripheral amoebic populations were replated on NATEin methane, bacterial growth--presumably methanotrophs--sometimesreoccurred. In addition, when amoebic populations grown on NNAE plateswere stored for several weeks such that encystation occurred, viablemethanotrophic and heterotrophic bacteria were still present as evidentfrom colony growth when the amoebic cysts were transferred to NATE mediain a methane atmosphere. When consortia, maintained on NATE in methaneand air, were filtered through 0.8 μm filters and the filtrate replatedon NATE in methane, methanotrophic and heterotrophic colonies free ofdetectable amoebae were occasionally obtained.

Thus, we have been able to free heterotrophic bacteria frommethanotrophic bacteria and amoebae and have been able to free theamoebae from methanotrophic and heterotrophic bacteria. We have not asyet, however, been able to disassociate the methanotrophic from theheterotrophic bacteria following separation from the amoebae.

                  TABLE 1                                                         ______________________________________                                        Characterization of Bacteria from Consortium 14                               Possible    Growth on Methane                                                                           Unique                                              Identification                                                                            as Sole Carbon                                                                              Characteristics                                     ______________________________________                                        Pseudomonas sp.                                                                           +             Catalase 3+                                         Pseudomonas sp.                                                                           +             -                                                   Bacillus sp.                                                                              +             Gelatin +                                           Acinetobacter sp.                                                                         +             -                                                   Bacillus sp.                                                                              -             Orange pigmentation                                 Acinetobacter sp.                                                                         -             -                                                   ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Characterization of Bacteria from Consortium 27P                              Possible    Growth on Methane                                                                           Unique                                              Identification                                                                            as Sole Carbon                                                                              Characteristics                                     ______________________________________                                        Pseudomonas sp.                                                                           +             Catalase 3+                                         Pseudomonas sp.                                                                           +             -                                                   Bacillus sp.                                                                              -             Orange pigmentation                                 Bacillus sp.                                                                              +             Gelatin +                                           Acinetobacter sp.                                                                         -             -                                                   Pseudomonas sp.                                                                           -             -                                                   Xanthomonas sp.                                                                           -             Oxidase -                                           ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Characterization of Bacteria from Consortium 46                               Possible     Growth on Methane                                                                            Unique                                            Identification                                                                             as Sole Carbon Characteristics                                   ______________________________________                                        Pseudomonas sp.                                                                            +              -                                                 Paracoccus sp.                                                                             -              ONPG +                                            Pseudomonas sp.                                                                            +              Vacuoles                                          Moraxella sp.                                                                              +              ONPG +                                            Pseudomonas sp.                                                                            +              Nitrate +                                         Cytophaga sp.                                                                              +              Corrodes Agar                                                                 (Possibly breaks                                                              C--C bonds)                                       Pseudomonas sp.                                                                            +              -                                                 Pseudomonas sp.                                                                            -              -                                                 Pseudomonas sp.                                                                            -              -                                                 Alcaligenes sp.                                                                            -              Citrate +                                         Hyphomicrobium sp.                                                            ______________________________________                                    

Electron microscopic examination of the consortia and themethanotroph/heterotroph mixture showed no evidence of a Type Imethanotroph which had been previously isolated from the aforementionedsite. Instead, examination of the methanotroph/heterotroph mixtureshowed some bacteria with typical gram-negative morphology and manycells with morphologic characteristics of Hyphomicrobium. Typicalfree-living amoebae with the expected "bulls-eye" nucleolus wereevident. Bacteria were commonly seen in the amoebic cytoplasm both inthe trophozoite and cyst stage. In both conditions the bacteria were inmembrane bound vacuoles.

The nature of these stable amoebae/bacteria associations appear to besomewhat symbiotic, indicated by the continuing presence of both amoebaeand heterotrophs on a minimal salts medium in a methane/air atmosphere.Neither heterotrophs nor amoebae alone are generally believed to persistunder these conditions. Indeed, the heterotrophs and amoebae isolatedfrom the consortia could no longer grow under such conditions. The mostlikely explanation of the stability of the amoebic-bacteria consortia onNATE in a methane atmosphere is the growth of the methanotrophicbacteria allowing the persistance of the amoebae and heterotrophs.

The electron microscopic examination of the consortia andmethanotroph/heterotroph mixtures also helped explain some of thecultural observations and difficulties. In light of the observedintra-amoebic presence of bacteria, it is seen why the initial isolationof amoebic cultures yielded bacterial isolates on subsequent transfer ofthe amoebae to a methane in air environment. It also explains thedifficulties in trying to free the amoebae of the methanotrophs andheterotrophs. Similarly, the difficult, and as yet unrealized,separation of methanotrophs and heterotrophs is likely explained by theabundance of Hyphomicrobium which most likely makes up the majority ofthe bacterial component. This genera of microorganism is noted for itsproclivity for associating with other microorganisms such that theirisolation in pure culture has been rarely attained. Hyphomicrobium cangrow in the presence of single-carbon sources and mineral salts as usedherein. They can be found in association with methanotrophs and maydegrade methanol produced by methanotrophs, preventing the toxicaccumulation of methanol. Whether the Hyphomicrobium degrade TCE is notpresently testable since we have not as yet separated them from theother bacteria in spite of trying density gradients and othertechniques.

The amoebae in the trophozoite or cyst form may provide a more stableniche for the metabolic activities of the associated methanotrophs andheterotrophs. For instance, the ability of free-living amoebae toprotect associated bacteria from the killing effects of chlorine hasbeen demonstrated. Thus, the isolation of the methanotrophs fromencysted and subsequently excysted amoebae may be pertinent.

Type I methanotrophs could not be identified by either electronmicroscopy or by culture from the amoebae/bacteria consortia describedherein. However, degradation of TCE by the total consortia, but not byheterotrophs or amoebae populations per se, suggests the methanotrophicbacteria are the TCE-degrading component of the consortia. Unlike thedegradation of TCE by Type I methanotroph isolated by from the samesite, a greater proportion of the ¹⁴ C was found in CO₂ as opposed tocellular and soluble fractions when TCE was degraded by the consortiadescribed herein.

EXAMPLE I

The ability of the consortia to degrade TCE in a methane atmosphere wastested. The microorganisms were incubated in 100 mL of liquid NATEmedium in 250 mL bottles fitted with teflon septa. The test bottles wereinjected with 12 mL of filtered sterilized methane (0.536 mmol) in 150mL of headspace volume. They were supplemented with [1,2-¹⁴ C]trichloroethylene (3.0 mCi/mmol [111 MBq/mmol], 95% pure by GC,Pathfinder Laboratories, St. Louis, Mo.). The test bottles were invertedand incubated for 12-14 days at 22°-24° C. on a shaker platform. Thecontents were analyzed for TCE periodically. Autoclaved cultures wereused as negative controls. Other controls included incubating culturesin air without methane and testing of E. coli in place of the consortia.After incubation, the content of ¹⁴ C-TCE in cellular material, CO₂, andculture fluid was determined by conventional scintillation techniques.TCE was degraded by the various consortia as indicated in the fate of ¹⁴C-labelled TCE exposed to the consortia in a methane in air atmosphere,shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        Degradation of .sup.14 C-Trichloroethylene (TCE) by Microbial                 Consortia Average percent .sup.14 C per fraction.sup.b                                No. of                                                                Consortia.sup.a                                                                       Expts.   Cell Pellet                                                                              CO.sub.2 Soluble                                  ______________________________________                                        14      2        13 (0-25)  87 (75-100)                                                                             0                                       27B     3        11 (6-16)  49 (25-89)                                                                             40 (0-69)                                27P     2        11         73 (57-89)                                                                             16 (0-32)                                46      5        10 (0-19)  75 (50-100)                                                                            15 (0-37)                                ______________________________________                                         .sup.a Numerical designation indicates well water of origin.                  .sup.b Determined after subtracting E. coli or autoclaved controls which      were about 10% of that observed with consortia. Percent of .sup.14 CTCE       degraded by the consortia ranged from 30-40% after 12 days incubation at      25° C.                                                            

EXAMPLE II

Amoebae and bacterial heterotrophs from the consortia were tested forand found not to have ability to degrade TCE. Mixtures of methanotrophicand heterotrophic bacteria isolated from the consortia degraded TCE.Neither the heterotrophic bacteria nor amoebae population withoutassociated methanotrophic bacteria degraded TCE. Trichloroethylene wasnot appreciably degraded by consortia or methanotrophs in air. Neitherautoclaved consortia nor E. coli degraded appreciable amounts of ¹⁴C-TCE. Results are shown in Table 5.

                  TABLE 5                                                         ______________________________________                                        Degradation of .sup.14 C-Trichloroethylene (TCE) by Components                of Microbial Consortia Counts of .sup.14 C per Fraction.sup.a                 Consortium  No. of                                                            Component   Expts.   Cell Pellet                                                                             CO.sub.2                                                                             Soluble                                 ______________________________________                                        Original consortium                                                                       2        656       2844   2010                                    Methanotrophs/                                                                            2        854       3037   2185                                    Heterotrophs.sup.b                                                            Amoebae.sup.c                                                                             2        142       145    570                                     Heterothrophs.sup.d                                                                       2        129       105    600                                     E. coli control                                                                           2        129       113    550                                     ______________________________________                                         .sup.a Average total counts added to test system was 12,875. Consortia,       amoebae, methanotrophs and heterotrophs incubated with TCE in air in the      absence of methane showed counts similar to E. coli control.                  .sup.b The original consortium was filtered through a 0.8 μm filter an     passaged three times on a mineral salts media in a methane in air             atmosphere. When the resultant microbial growth was tested for amoebae an     heterotrophs no amoebae were detected but heterotprohs were still present     .sup.c Amoebae were passaged three times on nonnutrient agar spread with      lawn of E. coli. When replanted on mineral salts in a methane atmosphere      no bacterial growth was apparent.                                             .sup.d Heterotrophs were passaged three times on TSA and were free of         amoebae and methanotrophs.                                               

Because of the successful degradation of TCE by two of thebacteria/amoebic consortia (27, 46), these consortia were also testedfor their ability to alter or degrade 2,4,6 trinitrotoluene (TNT).

EXAMPLE III

Sterile NATE solution was mixed in equal volume with a saturated TNTsolution (100 mg/L) for a total volume of 100 ml and inoculated with10¹² organisms. This was done in 250 ml teflon sealed bottles with 15 mlof methane added to the headspace through the teflon septa. Thesebottles were tested under the following conditions: 46consortium+methane, 46 consortium-methane, 27

consortium+methane, 27

consortium-methane, TNT solution+methane (no bacteria) and TNT solution(no methane, no bacteria).

No visible differences were seen in any of the bottles except for the 46consortium with methane in which the liquid medium turned yellow. Thecolor change occurred rapidly (overnight) on approximately Day 12 inrepeat experiments. The media from each bottle was analyzed by highpressure liquid chromatography (HPLC) after 3 weeks incubation; the 46consortium under methane showed a decrease in TNT levels.

EXAMPLE IV

In an experiment similar to Example III using ¹⁴ C-labeled TNT, the ¹⁴C-TNT was associated with the cell pellet, and was not extractable withacetonitrile (unlike the other bottles). This suggests that the TNT istightly associated with the cell, or has become nonpolar since TNT andits metabolites are readily extractable with acetonitrile. Table 6 showsthat "altered" TNT is, in fact, associated with the cell pellet and hasnot been degraded to carbon dioxide nor remained in the supernate.

                  TABLE 6                                                         ______________________________________                                        .sup.14 C-TNT Alteration by 46 Consortium                                                 Bottle #1                                                                              Bottle #2                                                                              Control                                         ______________________________________                                        .sup.14 CO.sub.2                                                                             1.5%       1.9%    4.1%                                        .sup.14 TNT in supernate                                                                    28.9%      31.7%    89.8%                                       .sup.14 TNT in cell pellet                                                                  69.6%      66.4%    3.5%                                        ______________________________________                                    

An organism from Consortium 46, Cytophaga sp., was observed to decomposeagar (i.e. break C--C bonds), prompting experiments to test isolatedorganisms' ability to alter, degrade, or disperse C--C basedhydrocarbons, especially nonpolar substances such as creosote, oil, waxand grease. Oil, creosote, wax, and grease can be dispersed intomicrodroplets in order to facilitate degradation by consortia,additional organisms, or other methods or natural processes.

EXAMPLE V

Dilute suspensions of each organism in Consortium 46 were placed,respectively, in 100 ml saline with 0.1 gm of creosote, and in 100 mlsaline with 0.1 gm of crude oil. Five organisms were found to have theability to immediately disperse the creosote and crude oil intomicrodroplets. It was found that the five organisms represented twodifferent modes of action. Two of the bacteria, isolates T and 9, werefound to have this ability only if the cells were present. The otherthree organisms, isolates 13, 15, and 1s, were found to produce adispersant which produced the dispersion effect. In all cases thedispersant was more effective after autoclaving and resultantsterilization. Each dispersant greatly reduced the liquid-liquidinterstitial surface tension, indicated by tensiometer tests. Samples ofwax and grease were also dispersed. Isolate 13 was found to beespecially effective in dispersing wax.

EXAMPLE VI

The bacterial isolates described in Example V were tested on woodcontaminated with creosote, resulting in obvious desorption of creosotefrom the wood.

Soil fines, postulated to hinder decomposition of contaminatingsubstances by creating pockets which can not be reached by bacteria orfungi, can be eliminated.

EXAMPLE VII

The bacterial isolates described in Example V were tested on soilcontaminated with creosote. The dispersants altered the physical natureof the soils by virtually eliminating the fines, or very fineparticulate matter.

Additional testing with various organic compounds such as nicotineshowed that the dispersant allowed maximal extraction in a much shortertime frame than expected, because of its ability to microemulsifynonpolar organic compounds.

Preliminary analysis by column chromatography and concentrationprocedures showed that the dispersant produced was slightly different ineach bacteria (different molecular weights and effectiveness). Eachdispersant appears to have a negative charge associated with at least apart of the molecular structure. The dispersants are assumed to bepartly denatured protein and possibly partly lipid in nature sincelipase can significantly reduce their effectiveness. These appear to bethe first bacterially produced dispersants or microemulsifiers known toexist.

Optimization of growth conditions for three of the five organisms hasbeen performed and these organisms can be rapidly grown on standardmicrobiological media under slightly different temperatures and pHconditions.

Many types of bacteria have also been found to be able to use theseproducts as a nutrient base which has the added advantage of not onlydispersing C--C based compounds, but simultaneously fertilizing theindigenous flora which could facilitate decomposition.

Additionally, it has been found that all organisms isolated from theconsortia which can disperse oil, creosote, wax, and grease also havethe ability to precipitate iron added to the growth medium.

Bacteria capable of dispersing oil, creosote, wax, and grease werecharacterized as follows:

Isolate 15: Family Pseudomonadaceae, Genus Pseudomonas Species probablypseudoalcaligenes: Gram negative rod, 0.5-1.5 um. Oxidase negative,catalase positive, motile, non sporulating, TSI (K/-). Colony morphologyon Tryptic Soy Agar media-translucent, undulate, diffuse margins,mucoid, pale white-yellow green. Produces green diffusible pigment onMueller-Hinton agar with grape-like odor. Colonies on BCYE medium haveirregular margins are mucoid and yellow-white. Aerobic, but grows weaklyin methane atmosphere as sole carbon source. Can use the followingcarbon sources for growth and metabolism: tween 40, tween 80,N-acetyl-D-glucosamine, L-arabinose, D-arabitol, D-fructose,D-galactose, D-mannitol, D-mannose, D-trehalose, methyl pyruvate,mono-methyl succinate, acetic, formic, citric, lactic, valeric,propionic and succinic acids, glycerol, serine, ornithine, leucine,histidine and alanine. ONPG, VP, nitrate reduction and gelatinenegative. Does not possess arginine dihydrolase, lysine decarboxylase,urease, tryptophane deaminase or ornithine decarboxylase. Hydrogensulfide is not produced. Optimum temperature and pH for growth are 20°C. and 7.0, respectively.

Isolate 13: Family (uncertain), Genus Alcaligenes, Species (unknown):Gram negative rod, 0.5-1.5 um. Oxidase positive, catalase positive,motile, non sporulating, TSI (K/-). Colony morphology on Tryptic SoyAgar media-opaque, entire, dry, off-white with a musty odor. Growth onMueller-Hinton agar is sparse with small colonies that are translucent,entire and pale yellow. On BCYE medium the colonies are small, entire,pale-yellow with a musty odor. Aerobic-does not grow in methaneatmosphere as sole carbon source. Can use the following carbon sourcesfor growth and metabolism: tween 40, tween 80, L-arabinose, psicose,D-fructose, methyl pyruvate, mono-methyl succinate, acetic, gluconic,formic, citric, lactic, valeric, propionic and succinic acids, glycerol,serine, leucine, histidine and alanine. ONPG, VP, nitrate reduction andgelatine negative. Does not possess arginine dihydrolase, lysinedecarboxylase, urease, tryptophane deaminase or ornithine decarboxylase.Hydrogen sulfide is not produced. Citrate positive. Optimum temperatureand pH for growth are 25° C. and 7.5, respectively.

Isolate 1s: Family Pseudomonadaceae, Genus Pseudomonas, Species(unknown): Gram negative rod, 0.5-1.5 um. Oxidase positive, catalasepositive, motile, non sporulating, TSI (K/-). Colony morphology onTryptic Soy Agar, BCYE and Mueller-Hinton-translucent, entire, mucoid,pale white to yellow with a faint grape-like odor. Aerobic, but growsweakly in methane atmosphere as sole carbon source. Can use thefollowing carbon sources for growth and metabolism: tween 40, tween 80,N-acetyl-D-glucosamine, L-arabinose, methyl pyruvate, monomethylsuccinate, acetic, citric, itaconic, glutaric, lactic, propionic,sebacic, aspartic and succinic acids, L-proline, L-phenyl alanine andphenyl ethylamine. ONPG, VP, nitrate reduction and gelatine negative.Does not possess arginine dihydrolase, lysine decarboxylase, urease,tryptophane deaminase or ornithine decarboxylase. Hydrogen sulfide isnot produced. Optimum temperature and pH for growth are 25° C. and 7.0,respectively.

Isolate T: Family Neisseriaceae, Genus Acinetobacter, Species (unknown):Gram negative coccobacillus, 0.5-1.0 um. Oxidase negative, catalasepositive, non motile, non sporulating, TSI (K/-). Colony morphology onTryptic Soy Agar media-opaque, entire, dry, white. Aerobic, but growsweakly in methane atmosphere as sole carbon source. ONPG, VP, nitratereduction and gelatine negative. Does not possess arginine dihydrolase,lysine decarboxylase, urease, tryptophane deaminase or ornithinedecarboxylase. Hydrogen sulfide is not produced. Optimum temperature andpH for growth are 25° C. and 7.0, respectively.

Isolate 9: Family Pseudomonadaceae, Genus Pseudomonas, Species(unknown): Gram negative rod, 0.5-1.5 um. Oxidase negative, catalasepositive, motile, non sporulating, TSI (K/-). Colony morphology onTryptic Soy Agar media-opaque, entire, dry, yellow. Aerobic, but growsweakly in methane atmosphere as sole carbon source. ONPG, VP, nitratereduction and gelatine negative. Does not possess arginine dihydrolase,lysine decarboxylase, urease, tryptophane deaminase or ornithinedecarboxylase. Hydrogen sulfide is not produced. Optimum temperature andpH for growth are 25° C. and 7.0, respectively.

Some of the bacterial isolates from the consortium showed antibiotic(antimicrobial and/or antifungal) activity.

EXAMPLE VIII

Tests were carried out using a standard double blind protocol whereinisolates inhibited fungal growth in liquid media. In this protocol,individual bacteria from the consortia and various, unidentified testfungi were placed together in dilute media concentrations in the wellsof a microtiter plate. The microtiter plate was incubated for one weekand then examined microscopically for the presence of either bacteria,fungi or both. Control wells established the viability of all organisms.Fungi were not amoeba derived and represented common environmentalfungi; therefore bacterial inhibition of the fungi is stressed as animportant advantage of the present invention. If bacteria were presentand no fungi grew, this was interpreted as the bacteria possessing anantifungal agent. If fungi were present and no bacteria, this wasinterpreted as the fungi possessing an antibacterial agent. If bothbacteria and fungi were present, no antibiotic effects were detected.Since the bacteria were originally amoeba associated, a contribution ofthe amoeba to antimicrobial activity of the bacteria cannot be ruledout. Results are shown in Table 7.

                                      TABLE 7                                     __________________________________________________________________________    Bacterial isolates from consortia 46 and 27P                                  FUNGUS                                                                              A B C D1                                                                              D2                                                                              G H I K1                                                                              K2                                                                              L1                                                                              M N O P1                                                                              P2                                                                              Q R S T AF 1S                                                                              13                         __________________________________________________________________________    1     - - - - - - - - - - - - - - - - - - - - -  - -                          2     # # # - - - # # - - - - - - - - - #        -                                                                             -                                                                             -                                                                             - -                          3     # # # - - - # # - - - - - - - - - #        -                                                                             -                                                                             -                                                                             - -                          4     - - # - - + # - + + + + + + + - + #        -                                                                             -                                                                             +                                                                             + +                          5     - - # + - + - - + + + + + + + + + #        -                                                                             -                                                                             +                                                                             + +                          6     - - - - - - - - - - - - - - - - - #        #                                                                             -                                                                             -                                                                             - -                          7     - - - - - - - - - - - - - - - - - -        -                                                                             -                                                                             +                                                                             - -                          8     - - - - - - - - - - - - - - - - - -        -                                                                             -                                                                             -                                                                             + -                          9     - - - - - - - - + + + - - - - - - -        -                                                                             -                                                                             -                                                                             - -                          __________________________________________________________________________     + bacteria inhibited fungus                                                   # fungus inhibited bacteria                                                   - indicates no effect on either bacteria or fungus                       

Based on the ability of these consortia to degrade, disperse or altervarious compounds, these protozoan derived consortia are useful forbioremediation efforts. Pathogenicity testing of these consortia showedthat no known pathogens are present, enhancing their usefulness.

Bioremediation is generally carried out by utilizing conventionalmethods. These include the use of bioreactors, and fertilization orcomposting, allowing indigenous organisms to remove the contaminants.The subject consortia can be utilized whole or in part for thesemethods.

Bioreactors are typically placed on-site and require moving contaminatedmaterial from the site of contamination to the bioreactor forprocessing. The bioreactor itself can consist of various shapes andsizes but is typically a cylinder which is filled with inert materialwhich bacteria can attach to. As the contaminated material is added tothe reactor, the large surface area created by the inert material allowsmaximal contact of the contaminant with the bacteria responsible fordegrading it. Our consortia are ideally suited for this type ofremediation and can be added directly onto the inert material.Concentrations which would be effective range from 1×10¹⁰ to 1×10¹²organisms per liter of bioreactor surface area.

Fertilization typically entails adding compounds (nitrogen, phosphate)to contaminated soils or other contaminated substances in order tostimulate indigenous organisms which may be capable of degrading thecontaminant to less hazards concentrations or altering it to non-toxicforms. This is usually performed over large areas at the site ofcontamination and is usually sprayed over the area so that it soaks intothe ground. This process, without the addition of degrading bacteria,typically requires long time periods and is not totally effective.Addition of our consortia would enhance this procedure sincefertilization may also stimulate some of the components of our consortiaas well as indigenous organisms. Also, the dispersants produced by theconsortia may help in desorbing some contaminants and, by eliminatingsoil fines, should enhance the degradative ability of both indigenousand added microbes. Our consortia could yield itself quite readily tothis type of bioremediation and could be added directly to thefertilizer if the concentrations are not too high. Effectiveconcentrations would range from 1×10¹⁰ to 1×10¹² organisms per liter ofspray solution.

From years of manufacturing and handling of explosive materials at U.S.Army facilities, over 1,000,000 cubic yards of soil and sediments havebecome contaminated with TNT and other explosives. The only availableremedial option is incineration which can be very expensive. Compostingis currently being tested as an alternative remediation technology whenincineration is ruled out. This is accomplished by mixing contaminatedsoil with manure, wood chips, hay, fertilizer and other amendments,creating a favorable environment for the indigenous microbes (from thecontaminated soils) enabling them to mineralize the explosives. Additionof our consortia to such a procedure would also supplement and increasethe rate at which these compounds are degraded. It could also beginalteration of TNT before indigenous organisms increase to such an extentas to be useful. In addition, alteration of TNT by consortia 46, couldproduce metabolites which are more easily degraded than TNT by otherorganisms. As previously discussed, the desorption of contaminants andthe elimination of soil fines could enhance the reaction. Effectiveconcentrations would range from 1×10¹⁰ to 1×10¹² organisms per kilogramof compost.

Deposit of Microorganisms

The applicant, in accordance with the provisions of the Budapest treatyon the international recognition of the deposit of microorganisms forthe purposes of patent procedure, did deposit samples of consortia 46,276, 27p, and 14 with the American Type Culture Collection (ATCC), 12301Parklawn Drive, Rockville, Md. 20852, U.S.A. Consortium 46 was depositedwith the ATCC on Oct. 11, 1990 and assigned ATCC deposit reference No.40908. Consortium 27p was deposited with the ATCC on Nov. 8, 1990 andassigned ATCC deposit reference No. 55120. Consortium 27b was depositedwith the ATCC on Jul. 16, 1992 and assigned ATCC deposit reference No.55343. Consortium 14 was deposited with the ATCC on Jul. 16, 1992 andassigned ATCC deposit reference No. 55342.

While there has been shown and described what is at present consideredthe preferred embodiment of the invention, it will be obvious to thoseskilled in the art that various changes and modifications may be madetherein without departing from the scope of the invention as defined bythe appended claims.

What is claimed is:
 1. A method of separating a heterotrophic bacteriumfrom a free living amoeba/bacteria consortium comprising:providing afree living amoeba/bacteria consortium including a heterotrophicbacterium, said amoeba/bacteria consortium being selected from the groupconsisting of ATCC 55120, ATCC 40908, ATCC 55342, ATCC 55343, a mutantof one of these consortia possessing all the identifying characteristicsof said one of these consortia and mixtures thereof; and placing saidamoeba/bacteria consortium in contact with a medium which is selectivefor said heterotrophic bacterium to separate said heterotrophicbacterium from said amoeba/bacteria consortium.
 2. The method accordingto claim 1 wherein said medium comprises trypticase soy agar.